Assessment of stereoselectivity of trimethylphenylalanine analogues of delta-opioid [D-Pen(2),D-Pen(5)]-enkephalin.

[D-Pen(2),D-Pen(5)]-Enkephalin (DPDPE) is an enzymatically stable delta-opioid receptor-selective peptide, which was modified by the trimethylation of the Phe(4) residue to give beta-methyl-2', 6'-dimethylphenylalanine (TMP), resulting in four conformations : (2R,3S)-beta-Phe-DPDPE, (2R,3R)-beta-Phe-DPDPE, (2R, 3S)-beta-Phe-DPDPE, and (2S,3R)-beta-Phe-DPDPE. Synthesis was by solid-phase techniques using enantiomerically pure amino acids to give the four optically pure diastereoisomer peptides. The potency and selectivity (delta- versus mu-opioid receptor) were evaluated by radioreceptor binding in rat brain, with a mu/delta ratio decrease for all TMP conformations, compared with the parent compound (DPDPE). Octanol/buffer distribution analysis showed enhanced lipophilicity of all TMP forms, with a sixfold enhancement associated with (2S,3S)-TMP. In situ vascular perfusion in anesthetized rats showed a 1.6-fold (p < 0.01) increase in the ratio of brain uptake for (2S,3S)-TMP and a 1.5-fold (p < 0.01) decrease in uptake for (2R,3R)-TMP. Saturability of (2S,3S)-TMP was shown (p < 0.01) against 100 microM unlabeled DPDPE, showing a shared nondiffusionary transport system. P-glycoprotein affinity was shown in situ for the parent and (2S,3S)-TMP (p < 0.01). Protein binding capacity of the TMP compounds in rat plasma and in situ mammalian bovine serum albumin-Ringer showed (2R,3S)-TMP and (2S,3R)-TMP with the lowest degree of protein binding (p < 0.01), and (2S,3S)-TMP and (2R,3R)-TMP with comparable affinities to DPDPE. Analgesia, via intravenous administration, showed significantly reduced (p < 0.01) end effect and time course for (2R,3R)-TMP, (2R,3S)-TMP, and (2S, 3R)-TMP as compared with DPDPE. These results demonstrate that topographical modification in a conformationally restricted peptide can significantly modulate potency and receptor selectivity, binding capacity, enzymatic stability, lipophilicity, P-glycoprotein affinity, and blood-brain barrier permeability, resulting in a change of bioavailability, and thereby provides insight for future peptide drug design.

Engineering of five 88-residue receptor-adhesive modular proteins containing a parallel alpha-helical coiled coil and two RGD ligand sites.

Several 88-residue proteins were designed, synthesized and examined as receptor-adhesive modular proteins (RAMPs). Three covalent and two noncovalent dimers were made from two 44-residue peptide chains containing three structural modules: RGD-A23a (ligand-spacer-coil) and A9a-RGD (coil-spacer-ligand). The ligand module contained the tripeptide Arg-Gly-Asp (RGD). The coil modules A9a and A23a were five-heptad alpha-helices engineered by Hodges and co-workers [Int. J. Peptide Protein Res. (1992) 40, 171-179]. By circular dichroic spectroscopy, each of these five RAMPs contained an alpha-helical coiled coil. The disulfide-bridged dimer RGD-A23a/RGD-A23a and its reduced form (RGD-A23a)2 had two N-terminal RGD sites. The disulfide-bridged dimer A9a-RGD/A9a-RGD and its reduced form (A9a-RGD)2 had two C-terminal RGD sites. However, the disulfide-bridged heterodimer RGD-A23a/A9a-RGD had one RGD site at each terminus with a 50 Angstrum coiled coil between them. The temperature at the midpoint of unfolding for each of the covalent homodimers RGD-A23a/RGD-A23a (67 degrees C) and A9a-RGD/A9a-RGD (69 degrees C) was slightly higher than that of the corresponding noncovalent homodimer (RGD-A23a)2 (62 degrees C) or (A9a-RGD)2 (68 degrees C) but much lower than that of the covalent heterodimer RGD-A23a/A9a-RGD (79 degrees C). The enthalpy and entropy of thermal unfolding were also significantly greater for the heterodimer than for the four homodimers, consistent with the heterodimer having the most stable coiled coil. Although the distance between its RGD sites was at least 50 Angstrum greater than that for the homodimers, this heterodimeric RAMP was only as active as the homodimers A9a-RGD/A9a-RGD and (A9a-RGD)2 in inhibiting the adhesion of A2058 melanoma cells to extracellular matrix proteins.(ABSTRACT TRUNCATED AT 250 WORDS)